Assay method and system for identification of P2Y receptor agonists and antagonists

Abstract

A method and system for assaying P2Y receptors. The method allows for direct detection of ligand binding events with a high signal to noise ratio. The ability to quantify binding events involving the P2Y receptor has heretofore been unavailable due to unavoidable non-selective binding of a radioligand or other traditional probes. A cell-free system for the study of P2Y receptors including a P2Y receptor; optionally, a protein that is normally associated with the P2Y receptor in nature; and a vesicle, is also disclosed.

What is claimed is:  
     
         1 . A method of screening candidate substances for an ability to modulate P2Y receptor-promoted biological activity, the method comprising: 
 (a) establishing a test sample comprising a substantially pure P2Y receptor;    (b) contacting the test sample with a candidate substance; and    (c) measuring an interaction, effect, or combination thereof, of the candidate substance on the test sample to thereby determine the ability of the candidate substance to modulate P2Y receptor-promoted biological activity.    
     
     
         2 . A method of screening candidate substances for an ability to modulate P2Y receptor-promoted biological activity, the method comprising: 
 (a) establishing replicate test and control samples that comprise a substantially pure biologically active P2Y receptor polypeptide;    (b) administering a candidate substance to test sample but not the control sample;    (c) measuring the activity of P2Y receptor-promoted biological activity in the test and the control samples; and    (d) determining that the candidate substance modulates P2Y receptor-promoted biological activity if a level of P2Y receptor-promoted activity measured for the test sample is greater or less than the level of P2Y receptor-promoted biological activity measured for the control sample.    
     
     
         3 . A method of screening candidate substances for an ability to modulate P2Y receptor-promoted biological activity, the method comprising: 
 (a) establishing a control system comprising a P2Y receptor and a ligand, wherein the P2Y receptor is capable of binding to the ligand;    (b) establishing a test system comprising a P2Y receptor, a ligand, and a candidate compound;    (c) measuring a binding affinity of a P2Y receptor and a ligand in the control and the test systems; and    (d) determining that the candidate compound modulates P2Y receptor-promoted activity in a cell-free system if the binding affinity measured for the test system is less than or greater than the binding affinity measured for the control system.    
     
     
         4 . The method of any of claims  1 ,  2  or  3 , wherein the method is carried out in a cell-free system.  
     
     
         5 . The method of  claim 4 , wherein a test sample or a control sample further comprises a vesicle comprising a P2Y receptor and a protein that normally interacts with a P2Y receptor in nature.  
     
     
         6 . The method of  claim 5 , wherein the P2Y receptor is selected from the group consisting of a P2Y 1  receptor, a P2Y 2  receptor, a P2Y 4  receptor, a P2Y 6  receptor, a P2Y 11  receptor and combinations thereof.  
     
     
         7 . The method of  claim 5 , wherein the protein that normally interacts with a P2Y receptor in nature is a G protein.  
     
     
         8 . The method of  claim 7 , wherein the G protein is selected from the group including but not limited to G a α, G q β, G a γ, G 11 α, G 12/13 α, G 12/13 β, G 12/13 γ, G i α, G i β, G i γ, G s α, G s ,β,G s γ, Gα 14 , Gα 16 , Gβγ dimers, and combinations thereof.  
     
     
         9 . The method of  claim 5 , wherein the protein that normally interacts with a P2Y receptor in nature is substantially pure.  
     
     
         10 . The method of  claim 5 , wherein a test sample or a control sample further comprises a ligand for the P2Y receptor or for the protein that normally interacts with a P2Y receptor in nature.  
     
     
         11 . The method of  claim 10 , wherein the ligand is selected from the group consisting NTP, NDP, a RGS protein, an agonist, an antagonist, and combinations thereof.  
     
     
         12 . The method of  claim 11 , wherein the RGS protein is selected from the group consisting of RGS1, RGS2, RGS4, RGS16 and combinations thereof.  
     
     
         13 . The method of  claim 10 , wherein the ligand, the P2Y receptor, the protein that normally interacts with a P2Y receptor, or combination thereof is detectably labeled.  
     
     
         14 . The method of  claim 13 , wherein the label is a radioactive moiety, a fluorescent moiety, a chemiluminescent moiety, or combination thereof.  
     
     
         15 . The method of  claim 14 , wherein the radioactive moiety is selected from the group consisting of  3 H,  32 P,  35 s,  14 C,  125 I and combinations thereof.  
     
     
         16 . The method of  claim 14 , wherein the fluorescent moiety is selected from the group consisting of a near-infrared fluorescent dye, dinitrophenyl, fluorescein and derivatives thereof, rhodamine, derivatives of rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, Texas red, Rhodamine green, Oregon green, Cascade blue, phycoerythrin, CY3, CY5, CY2, CY7, coumarin, infrared 40, MR 200, IRD 40, green fluorescent protein and combinations thereof.  
     
     
         17 . The method of  claim 14 , wherein the chemiluminescent moiety is selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester, luciferin, luciferase and aequorin and combinations thereof.  
     
     
         18 . The method of  claim 14 , wherein the binding affinity is assessed by comparing an amount of bound labeled ligand to an amount of unbound labeled ligand.  
     
     
         19 . The method of  claim 18 , wherein the bound and unbound labeled ligands are separated by contacting a test sample with a separation matrix.  
     
     
         20 . The method of  claim 19 , wherein the separation matrix comprises activated charcoal.  
     
     
         21 . The method of  claim 14 , wherein a detectable signal is generated from resonant interaction between two energy emitting label moieties.  
     
     
         22 . The method of any of claims  1 ,  2  or  3 , wherein the P2Y receptor-promoted biological activity is selected from the group consisting of hydrolysis of NTP molecules to NDP molecules, formation of NTP molecules from NDP molecules, modulation of intracellular calcium levels, modulation of phospholipase C activity, modulation of adenylate cyclase activity, translocation of RhoA to membranes, formation of a network of stress fibers, phosphorylation of myosin light chains, cell differentiation modulation of NTPase activity, shape change in platelets and combinations thereof.  
     
     
         23 . The method of any of claims  1 ,  2  or  3 , wherein the method is carried out in at least one well of a multi-well plate.  
     
     
         24 . The method of any of claims  1 ,  2  or  3 , further comprising screening a plurality of candidate substances simultaneously.  
     
     
         25 . The method of any of claims  24 , wherein the method is carried out in multiple wells of a multi-well plate.  
     
     
         26 . A cell-free system for the study of P2Y receptors comprising: 
 (a) a P2Y receptor;    (b) a protein that normally interacts with the P2Y receptor in nature; and    (c) a vesicle.    
     
     
         27 . The system of  claim 26 , wherein the P2Y receptor is substantially pure.  
     
     
         28 . The system of  claim 26 , wherein the P2Y receptor is selected from the group consisting of a P2Y 1  receptor, a P2Y 2  receptor, a P2Y 4  receptor, a P2Y 6  receptor, a P2Y 11  receptor and combinations thereof.  
     
     
         29 . The system of  claim 26 , wherein the protein that normally interacts with a P2Y receptor in nature is substantially pure.  
     
     
         30 . The system of  claim 26 , wherein the protein that normally interacts with a P2Y receptor in nature is a G protein.  
     
     
         31 . The system of  claim 30 , wherein the G protein is selected from the group including but not limited to G q α, G q β, G q γ, G 11 α, G 12/13 α, G 12/13 β, G 12/13 γ, G i α, G i β, G i γ, G s α, G s β,G s γ, G α   14 , Gα 16 , Gβγ dimers, and combinations thereof.  
     
     
         32 . The system of  claim 26 , further comprising a ligand for the P2Y receptor or for the protein that normally interacts with a P2Y receptor in nature.  
     
     
         33 . The system of  claim 32 , wherein the ligand is selected from the group consisting NTP, NDP, a RGS protein, an agonist. an antagonist, and combinations thereof.  
     
     
         34 . The system of  claim 33 , wherein the RGS protein is selected from the group consisting of RGS1, RGS2, RGS4, RGS16 and combinations thereof.  
     
     
         35 . The system of  claim 32 , wherein the ligand, the P2Y receptor, the protein that normally interacts with a P2Y receptor, or combination thereof is detectably labeled.  
     
     
         36 . The system of  claim 35 , wherein the label is a radioactive moiety, a fluorescent moiety, a chemiluminescent moiety, or combination thereof.  
     
     
         37 . The system of  claim 36 , wherein the radioactive moiety is selected from the group consisting of  3 H,  32 P,  35 S,  14 C,  125 I and combinations thereof.  
     
     
         38 . The system of  claim 36 , wherein the fluorescent moiety is selected from the group consisting of a near-infrared fluorescent dye, dinitrophenyl, fluorescein and derivatives thereof, rhodamine, derivatives of rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, Texas red, Rhodamine green, Oregon green, Cascade blue, phycoerythrin, CY3, CY5, CY2, CY7, coumarin, infrared 40, MR 200, IRD 40, green fluorescent protein and combinations thereof.  
     
     
         39 . The system of  claim 36 , wherein the chemiluminescent moiety is selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester, luciferin, luciferase and aequorin and combinations thereof.  
     
     
         40 . A method of producing a cell-free system for the assay of P2Y receptor-promoted activity, the method comprising: 
 (a) purifying a P2Y receptor;    (b) purifying at least one protein that normally interacts with the P2Y receptor in nature;    (c) reconstituting the P2Y receptor into a vesicle; and    (d) reconstituting at least one protein that normally interacts with the P2Y receptor in nature into the vesicle to thereby produce the cell-free system.    
     
     
         41 . The method of  claim 40 , wherein the P2Y receptor is selected from the group consisting of a P2Y 1  receptor, a P2Y 2  receptor, a P2Y 4  receptor, a P2Y 6  receptor, a P2Y 11  receptor and combinations thereof.  
     
     
         42 . The method of  claim 40 , wherein the P2Y receptor is expressed in an in vivo or in vitro expression system.  
     
     
         43 . The method of  claim 41 , wherein the expression system further comprises a recombinant vector comprising a nucleic acid sequence encoding a P2Y receptor.  
     
     
         44 . The method of  claim 43 , wherein the recombinant vector further comprises: 
 (a) a sequence of genomic viral DNA showing affinity for a host cell and possessing the ability to infect said host cell;    (b) a nucleic acid sequence encoding a P2Y receptor operatively linked to the sequence of genomic viral DNA, wherein the operatively-linked P2Y receptor is expressed in said host cell following infection of the cell; and    (c) a selectable marker.    
     
     
         45 . The method of  claim 44 , wherein the sequence of genomic viral DNA is baculoviral DNA.  
     
     
         46 . The method of  claim 43 , further comprising transfecting a cell with the recombinant vector under conditions suitable for the expression of the P2Y receptor to thereby produce a P2Y receptor.  
     
     
         47 . The method of  claim 46 , wherein the cell is a prokaryotic cell or is a eukaryotic cell.  
     
     
         48 . The method of  claim 47 , wherein the cell is an insect cell.  
     
     
         49 . The method of  claim 42 , wherein the P2Y receptor has a sequence of at least six histidine residues at the N-terminal end of the P2Y receptor protein.  
     
     
         50 . The method of  claim 42 , wherein the P2Y receptor has a sequence of at least six histidine residues at the C-terminal end of the P2Y receptor protein.  
     
     
         51 . The method of  claim 42 , wherein the P2Y receptor comprises a FLAG® epitope at the N terminal end of the P2Y receptor protein.  
     
     
         52 . The method of  claim 42 , wherein the P2Y receptor expresses a FLAG® epitope at the C terminal end of the P2Y receptor protein.  
     
     
         53 . The method of  claim 49  or  50 , wherein the P2Y receptor is purified by passing the receptor over a residue comprising nickel atoms.  
     
     
         54 . The method of  claim 51  or  52 , wherein the P2Y receptor is purified by binding the receptor to a detectable anti-FLAG® antibody and isolating the complex.  
     
     
         55 . The method of  claim 40 , wherein the proteins that normally interact with the P2Y receptor in nature are expressed in an in vitro or in vivo expression system.  
     
     
         56 . The method of  claim 55 , wherein the protein that normally interacts with a P2Y receptor in nature is a G protein.  
     
     
         57 . The method of  claim 7 , wherein the G protein is selected from the group including but not limited to G q α, G q β, G q γ, G 11 α, G 12/13 α, G 12/13 β, G 12/13 γ, G i α, G i β, G i γ, G s α, G s β,G s γ, Gβγ dimers, and combinations thereof.  
     
     
         58 . The method of  claim 40 , further comprising adding a ligand to the cell-free system.  
     
     
         59 . The method of  claim 58 , wherein the ligand is selected from the group consisting NTP, NDP, a RGS protein, an agonist, an antagonist, and combinations thereof.  
     
     
         60 . The method of  claim 59 , wherein the RGS protein is selected from the group consisting of RGS1, RGS2, RGS4, RGS16 and combinations thereof.  
     
     
         61 . The method of  claim 58 , wherein the ligand, the P2Y receptor, the protein that normally interacts with a P2Y receptor, or combination thereof is detectably labeled.  
     
     
         62 . The method of  claim 61 , wherein the label is a radioactive moiety, a fluorescent moiety, a chemiluminescent moiety, or combination thereof.  
     
     
         63 . The method of  claim 62 , wherein the radioactive moiety is selected from the group consisting of  3 H,  32 P,  35 s,  14 C,  125 I and combinations thereof.  
     
     
         64 . The method of  claim 62 , wherein the fluorescent moiety is selected from the group consisting of a near-infrared fluorescent dye, dinitrophenyl, fluorescein and derivatives thereof, rhodamine, derivatives of rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, Texas red, Rhodamine green, Oregon green, Cascade blue, phycoerythrin, CY3, CY5, CY2, CY7, coumarin, infrared 40, MR 200, IRD 40, green fluorescent protein and combinations thereof.  
     
     
         65 . The method of  claim 62 , wherein the chemiluminescent moiety is selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester, luciferin, luciferase and aequorin and combinations thereof.  
     
     
         66 . A method of producing a cell-free system for the assay of P2Y receptor-promoted activity, the method comprising: 
 (a) purifying a P2Y receptor; and    (b) reconstituting the P2Y receptor into a vesicle.    
     
     
         67 . A cell-free system for the study of P2Y receptors comprising: 
 (a) a P2Y receptor; and    (b) a vesicle.